Nitrones which are usable for the scavenging of free radicals

ABSTRACT

New nitrones which are usable for the scavaging of free radicals, are selected from 5-diethoxyphosphoryl-5-methyl-1-pyrroline 1-oxide, 5-phosphono-5-methyl-1-pyrroline 1-oxide, and 5-diethoxyphosphoryl-5-methyl-(2,3,3- 2  H 3 )-1-pyrroline 1-oxide, as well as its corresponding physiologically acceptable salts obtained by the action of an inorganic or organic base.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to cyclic nitrone derivatives which areusable as free-radical scavengers.

2. Description of the Related Art

A number of cyclic nitrones capable of scavenging free radicals areknown at the present time: 5,5-dimethylpyrroline N-oxide (DMPO) ismarketed in Europe by various companies such as Aldrich, Fluka andSigma. This compound, a free-radical trapping agent, is used only inelectron paramagnetic resonance experiments. Its application in thepharmaceutical and cosmetics fields is limited on account of itsinstability and its low solubility in biological media, as well as theinstability of its scavenging adducts.

M. J. Turner and G. M. Rosen describe, moreover, in their paperpublished in J.Med. Chem. 29 (12), 2439-2444 (1986), three cyclicnitrones which proved to be more effective free-radical scavengers thanDMPO, namely 5-butyl-5-methyl-1-pyrroline 1-oxide (BMPO),5,5-di-propyl-1-pyrroline 1-oxide (DPPO) and2-aza-2-cyclo-pentenespirocyclopentane 2-oxide (CPPO). However, thesolubility of these compounds in biological media is insufficient as theresult of their lipophilic properties, which are greatly superior tothose of DMPO.

SUMMARY OF THE INVENTION

New cyclic nitrones displaying great stability and better solubility inbiological media, and which lead to the formation of comparatively morestable scavenging adducts, have now been discovered.

The subject of the invention is cyclic nitrones of general formula I:##STR1## in which:

R₁ represents a phenyl or alternatively a (C₁ -C₁₈) alkyl,

R₂ represents a hydrogen or deuterium atom, a phenyl or (C₁ -C₁₈) alkylgroup or a radical Z of formula ##STR2## in which:

A is a single bond, a methylene group or an oxamethylene group (in whichthe oxygen atom is linked to the phosphorus atom of the radical Z), Yrepresents an oxygen atom and R represents a hydrogen atom, a (C₁ -C₁₈)alkyl or a (C₆ -C₁₈) aryl, or alternatively Y represents a methylenegroup and R represents a hydrogen atom, a (C₁ -C₁₇) alkyl or a (C₆ -C₁₈)aryl,

R₃, R₄ and R₅ represent, independently of one another, a hydrogen ordeuterium atom or a phenyl or (C₁ -C₁₈) alkyl group, and

R₆ represents a hydrogen or deuterium atom, a phenyl or (C₁ -C₁₈) alkylgroup or a radical ##STR3##

A representing a single bond and Y and R being as defined above,

R₇ represents a hydrogen or deuterium atom or a methyl group,

with the proviso that one and only one of the groups R₂ and R₆represents the radical Z of formula ##STR4##

A representing a single bond, a methylene group or an oxamethylene group(in which the oxygen atom is linked to the phosphorus atom of theradical Z), and R and Y being as defined above,

as well as their corresponding physiologically acceptable salts obtainedby the action of an inorganic or organic base.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

A preferred group of compounds of formula I consists of the compounds ofthe following formula II: ##STR5## in which:

R represents a hydrogen atom, a (C₁ -C₁₈) alkyl or a (C₆ -C₁₈) aryl,

R₁ represents a phenyl or alternatively a (C₁ -C₁₈) alkyl, R₃, R₄, R₅and R₆ represent, independently of one another, a hydrogen or deuteriumatom, a phenyl group or a (C₁ -C₁₈) alkyl, and

R₇ represents a hydrogen or deuterium atom or a methyl group,

Advantageously, R₁ represents a methyl group and R₇ is a hydrogen ordeuterium atom.

Among these preferred compounds, the following may be mentioned5-diethoxyphosphoryl-5-methyl-1-pyrroline 1-oxide (DEPMPO),5-phosphono-5-methyl-1-pyrroline 1-oxide, and5-diethoxyphosphoryl-5-methyl-(2,3,3-² H₃)-1-pyrroline 1-oxide(DEPMPO_(D)).

(C₁ -C₁₇) alkyl and (C₁ -C₁₈) alkyl groups are understood to mean groupshaving a linear or branched chain possessing from 1 to 17 carbon atomsor from 1 to 18 carbon atoms, respectively.

(C₆ -C₁₈) aryl group is understood to mean mono- or polycyclic aromaticcompounds comprising from 6 to 18 carbon atoms.

The physiologically acceptable salts are those formed by the action ofan inorganic or organic base on the compounds of formula I or II.

As preferred salts, the sodium salts of the phosphonic acids of formulaI or II may be mentioned.

The compounds of the invention may be prepared by a process whichconsists in oxidizing by means of a suitable oxidizing agent a compoundof general formula III: ##STR6## in which R₁, R₂, R₃, R₄, R₅, R₆ and R₇are as defined above.

In this reaction, the oxidizing agent can advantageously be an organicperoxy acid such as m-chloroper-benzoic acid, an inorganic oxidizingagent such as sodium tungstate or alternatively any combination ofinorganic oxide and hydrogen peroxide, such as selenium oxide in thepresence of hydrogen peroxide, or, as a further alternative, an organicoxidizing agent such as dimethyl-dioxirane.

The reaction conditions depend on the nature of the oxidizing agentselected, and it is within the capability of a person skilled in the artto determine them using his basic general body of knowledge. Thus, whenthe oxidation takes place by the action of an organic peroxy acid, aninert solvent such as, for example, dichloromethane or chloroform willbe chosen. When the oxidizing agent is hydrogen peroxide in aqueoussolution in the presence of an inorganic oxide, the selection will fallon a polar aprotic solvent such as, for example, acetone.

The temperature can vary in accordance with the nature of the compoundof formula III. It is advantageously between 0° C. and the refluxingtemperature of the solvent.

The compounds of general formula III in which R₂ represents the radical##STR7## where R and Y are as defined above, may be prepared by aprocess which consists in reacting a halo ketone of general formula IV:##STR8## in which X represents a halogen such as chlorine, bromine oriodine, and R₁, R₃, R₄, R₅, R₆ and R₇ are as defined above, with theproviso that the group R₆ does not represent the radical ##STR9## Arepresenting a single bond and R and Y being as defined above, with acompound of formula V: ##STR10## where Y and R have the same meaning asabove, in the presence of NH₃.

Here too, the reaction conditions depend on the nature of the reactantsof formula IV and V, and may be readily determined by a person skilledin the art. The reaction is generally conducted in a polar aproticsolvent. Advantageously, the solvent will be ethanol. The temperaturemay be between room temperature and the refluxing temperature of thesolvent.

The compounds of formulae IV and V are commercially available compounds,or compounds which can be prepared by a person skilled in the art usingknown methods.

The compounds of general formula III in which R₂ represents a radical##STR11## may be prepared by a process which consists in

a) reacting the corresponding compound of formula VI ##STR12## in whichR₂, R₃, R₄, R₅, R₆ and R₇ are as defined above, with the proviso thatthe group R₆ does not represent the radical ##STR13## A representing asingle bond and R and Y being as defined above, and Pr is a groupprotecting the amine function, such as, for example, a benzyloxycarbonylgroup, with a phosphorus derivative of formula P(YR)₃, where Y and R areas defined above,

b) and then deprotecting the secondary amine function by one of themethods known to a person skilled in the art.

The compounds of formula VI may be prepared in two steps from thefollowing compounds of formula VIII ##STR14## in which R₁ , R₃, R₄, R₅,R₆, R₇ and Pr are as defined for the compound VI.

In a first step, a compound of formula VIII is reacted with mercurydiacetate; the second step, which consists in treating the resultingproduct successively with potassium iodide and then iodine leadsdirectly to the corresponding compound of formula VI.

The compounds of formula VIII are readily prepared by a person skilledin the art using known methods, from commercially available compounds.

The compounds of general formula III in which R₂ represents a radical##STR15## may be prepared by a process which consists in:

a) reacting the corresponding compound of formula ##STR16## in which R₁,R₃, R₄, R₅, R₆, R₇ and Pr are as defined above, with the proviso thatthe group R₆ does not represent the radical ##STR17## A representing asingle bond and R and Y being as defined above, with a compound offormula X ##STR18## in which X represents a halogen such as chlorine andR and Y are as defined above,

b) and then deprotecting the secondary amine function by one of themethods known to a person skilled in the art.

The preparation of compounds of formula IX is carried out in two stepsfrom the corresponding compounds of formula VIII:

in a first stage the compound of formula VIII is reacted with a mercurydiacetate. In the second step, the resulting product is treated withoxygen in the presence of a hydride such as potassium borohydride toyield the expected compound of formula IX.

The preparation of the compounds of formula I in which R₆, represents aradical ##STR19## where R and Y are as defined above may be carried outin at least two different ways.

A first synthesis process consists in treating with sodium hydride thecorresponding compound of formula XI ##STR20## in which R₁, R₂, R₃, R₄,R₅, and R₇ are as defined above, with the proviso that R₂ does notrepresent a group ##STR21## R and Y being as defined above and Arepresenting a single bond, a methylene group or an oxamethylene group(in which the oxygen atom is linked to the phosphorus atom of theradical Z), and then in reacting the resulting sodium salt with acompound of formula X.

In the second synthesis process, a compound of formula XI is treatedsuccessively with N-bromo-succinimide and a compound of formula P(YR)₃in which Y and R are as defined above. When the compound of formula I inquestion is such that Y is an oxygen atom, this reaction is known by thename of the Arbuzov reaction.

The compounds of formula XI are prepared by means of an oxidizing agentfrom the corresponding compounds of formula III. The method used hasbeen explained in detail above.

The compounds of general formula I for which Y is an oxygen atom and Ris a hydrogen atom may be obtained from the corresponding compounds offormula I in which R is an alkyl, by the action of a trimethylsilylhalide such as trimethylsilyl bromide under anhydrous conditions,followed by aqueous hydrolysis. In this reaction, an aprotic inertsolvent may be used. Dichloromethane and chloroform may be mentioned aspreferred solvents. The temperature depends on the compound of formula Iin question, and may vary between room temperature and the refluxingtemperature of the solvent.

The compounds of formula I for which one or more of the radicals chosenfrom R₂, R₃, R₄, R₅, R₆ and R₇ represent a deuterium atom are preparedfrom the compounds of the same formula in which the deuterium atoms arereplaced by hydrogen atoms, by the action of sodium deuteroxide (NaOD)in heavy water (D₂ O). The reaction temperature depends on the nature ofthe compound of formula I in question. Temperatures between 0° C. andthe refluxing temperature of heavy water may be chosen with equalvalidity for this purpose.

The compounds of the invention are usable as free-radical scavengers. Assuch, and on account of their physicochemical properties, they findapplication in cosmetology and in the medical field.

In the human biological medium, several reactive oxygenated species,prooxidants of the O₂.⁻⁻, HO.sup.·, HOO.sup.· type, may formed from theoxygen transported in the body by respiration. In the normal situation,a low stationary concentration of these species is controlled by aseries of enzymatic or non-enzymatic antioxidants which effect theirremoval.

A situation of oxidative stress becomes established when there is alocal imbalance between antioxidants and prooxidants in favour of thelatter. Situations of oxidative stress have been demonstrated in thecase of various cardiovascular pathologies such as coronary ischaemia,arteriosclerosis and infarction, as well as in the case ofextracorporeal circulation employed in vascular surgery or alternativelyduring inflammatory, infectious or cell aging processes.

The compounds according to the invention are effective free-radicalscavengers which may be used in cosmetology as trapping agents of theprooxidant species responsible for cell aging.

In medicine, and more especially in the field of diagnosis, thecompounds according to the invention are useful in the evaluation ofoxidative stress. The direct detection of O₂.⁻⁻, HOO.sup.·, HO.sup.·free radicals generated in vivo is not possible by electron paramagneticresonance (EPR), in spite of their non-zero spin magnetism, as a resultof their instability: these radicals possess, in effect, a half-life ofthe order of 10⁻⁵ to 10⁻³ second.

Spin trapping is the technique used for detecting these radicals. Itsprinciple is as follows: the biological medium to be tested is placed inthe presence of a free-radical scavenger S. If free radicals such asO₂.⁻⁻, HO.sup.·, HOO.sup.· are present in the medium, they combine withthe scavenger S to form an adduct (S--O₂).⁻⁻, (S--OH).sup.· or(S--OOH).sup.·. This adduct is persistently paramagnetic, and can hencebe detected by electron paramagnetic resonance (EPR).

With respect to free radicals, the compounds according to the inventionhave proved to be effective scavengers permitting the detection by EPRof prooxidant free radicals in biological media.

The use of the compounds according to the invention as free-radicaltrapping agents or scavengers affords many advantages. The stability ofthese compounds and their solubility in biological media may bementioned.

The inventors have also been able to demonstrate that the kinetics ofscavenging of free radicals such as HOO.sup.· by the compounds of theinvention is very much faster than in the case of 5,5-dimethylpyrrolineN-oxide (DMPO) of the prior art.

The reaction of trapping of the HOO.sup.· radical by DMPO is slow andcharacterized by a, second order rate constant k, of 10M⁻¹ s⁻¹ ; the invivo formation of the superoxide radical is consequently difficult todetect with DMPO. In contrast, the rate of trapping of the HOO.sup.·radical by 5-diethoxyphosphoryl-5-methyl-1-pyrroline 1-oxide (DEPMPO), acompound according to the invention, is 2.5 times as fast.

Moreover, during the detection of HOO.sup.· with DMPO, several authorshave reported the decomposition of the (DMPO--OOH).sup.· adduct to the(DMPO--OH).sup.· adduct. This results in a poor reliability of spintrapping experiments carried out using DMPO.

On the other hand, in the case of DEPMPO, a compound according to theinvention, no decomposition of the (DEPMPO--OOH).sup.· adduct to the(DMPO--OH).sup.· adduct has been observed.

The stability of the adducts obtained from the compounds according tothe invention is further demonstrated by the following qualitative andquantitative experiments:

Freezing-thawing test

The possibility of storing blood samples taken from patients who are invarious situations which may involve the existence of oxidative stress(in cardiovascular surgery with extracorporeal circulation, or in thecase of myocardial infarction) is essential for the purpose of use inthe medical field. In the case of the compounds according to theinvention, the addition of the scavenging radical may be carried outbefore freezing and storage of the blood samples at 77 K as a result ofthe great stability of the scavenging adducts, as the followingexperiment shows:

The scavenging adducts of DEPMPO and the O₂.⁻⁻ and HOO.sup.· radicalswere analysed by electron paramagnetic resonance after storage at 77 Kand thawing. Their spectrum proved identical to that of the adductsbefore freezing, thereby demonstrating their good stability.

On the other hand, the same experiment using DMPO adducts lead to theappearance of many spurious signals interfering with the interpretationof the EPR spectra of the thawed adducts.

Kinetic study of the decomposition of nitrone-(O₂.⁻⁻ /HOO.sup.·) adducts

In this experiment, the radicals (O₂.⁻⁻ /HOO.sup.·) were produced byirradiation in the visible in the presence of the riboflavin (0.1mM)/DTPA (4 mM) system in 0.1M phosphate buffer (pH 7) at roomtemperature.

The nitrones studied are added to the irradiated solution at aconcentration of 0.08M.

When irradiation is stopped, the production of (O₂.⁻⁻ /HOO.sup.·)radicals ceases and the decrease in the nitrones-(O₂.⁻⁻ /HOO.sup.·)!adducts is monitored by EPR analysis.

The variation of the concentration of adducts with time was studied inthe case of the nitrones DMPO (of the prior art) and DEPMPO (of theinvention). The values of the concentration of adducts were recordedafter 2, 10, 20, 30 and 40 minutes. These values are shown in Table I.

                  TABLE I                                                         ______________________________________                                                  Concentration of                                                                            Concentration of                                      TIME      (DMPO)-(O.sub.2.sup..- /HOO.sup..)                                                          (DEPMPO)-(O.sub.2.sup..- /HOO.sup..)                  (Minutes) adducts       adducts                                               ______________________________________                                        0         100           100                                                   2         50            91                                                    10        0             68                                                    20        0             52                                                    30        0             42                                                    40        0             28                                                    ______________________________________                                    

The kinetics of disappearance of the adducts are of the first order inboth cases. The calculated rate constants are, respectively:

    k.sub.DMPO =1.4×10.sup.-2 s.sup.-1

    K.sub.DEPMPO =1.4×10.sup.-3 s.sup.-1

It emerges from the present study that the half-life of the(DEPMPO)-(HOO.sup.· /O₂.⁻⁻)! adducts is ten times as large as that ofthe (DMPO)-(HOO.sup.· /O₂.⁻⁻ ! adducts.

The subject of the invention is also a composition intended for use incosmetology, containing as active ingredient a compound according to theinvention: the products of general formula I are combined withexcipients, fragrances and appropriate colorants to form, for example,aerosols, solutions, creams or ointments.

Another subject of the invention is a diagnostic product which is usablein the evaluation of oxidative stress, comprising a compound accordingto the invention.

The examples which follow illustrate the invention without impliedlimitation. In the nuclear magnetic resonance (NMR) data, the chemicalshifts δ are expressed in ppm relative to TMS.

EXAMPLE 1 ##STR22## a) diethyl (2-methyl-2-pyrrolidinyl)phosphonate

A solution of 11.8 g of 5-chloro-2-pentanone and 14 g of diethylphosphite in 50 ml of ethanol is maintained at 50° C. and under a streamof ammonia for 4 hours. The reaction mixture is then filtered and thesolvent evaporated off. The residue is taken up with 40 ml of 2N HClsolution and then extracted with methylene chloride (2×50 ml). Theaqueous phase is neutralized by adding sodium bicarbonate and extractedwith chloroform. This organic phase is dried over sodium sulphate.Evaporation of the solvent under reduced pressure yields 13.7 g of acolourless oil.

b) 5-diethoxyphosphoryl-5-methyl-1-pyrroline 1-oxide

A solution of 4.4 g of 70% m-chloroperbenzoic acid in 40 ml ofchloroform is added dropwise to a solution, cooled to 0° C., of 2 g ofthe above pyrrolidine in 30 ml of chloroform. The reaction mixture isthen washed with saturated sodium bicarbonate solution (2×15 ml) andthen with saturated sodium chloride solution (15 ml). The organic phaseis dried over sodium sulphate and then concentrated under reducedpressure. The residue is chromatographed (silica, CH₂ Cl₂ /EtOH 85:15)and gives 0.4 g of a yellow oil.

EXAMPLE 2 ##STR23##

0.67 ml of trimethylsilyl bromide is added to 300 mg of5-diethoxyphosphoryl-5-methyl-1-pyrroline 1-oxide dissolved in 5 ml ofdry dichioromethane. The mixture is heated to reflux for 20 hours. Themethylene chloride is evaporated off under reduced pressure. The residueis hydrolysed with 0.8 ml of water in 6 ml of acetone. The precipitateis filtered off, washed with acetone (15 ml) and recrystallized indimethyl sulphoxide to give 137 mg of a white powder.

EXAMPLE 3 ##STR24## 12.06 g of 30% aqueous hydrogen peroxide solution isadded dropwise at 0° C. under argon to a solution of 8.61 g of5-diethoxyphosphoryl-2,5-dimethyl-1-pyrrolidine and 0.21 g of SeO₂ in 72ml of acetone. After the addition, the reaction mixture is left stirringat room temperature for 30 hours. The acetone is removed under reducedpressure. The aqueous phase is extracted with dichloromethane. Theorganic phase is dried over sodium sulphate and then concentrated underreduced pressure. The residue is chromatographed on a column (silica,CH₂ Cl₂ /EtoH 90:10) and yields 2.1 g of a yellow oil. EXAMPLE 4##STR25##

A solution of sodium deuteroxide (8N NaOD in D₂ O, 0.2 ml) in 10 ml ofheavy water, D₂ O, cooled beforehand, is added slowly to a solution of0.5 g of 5-diethoxyphosphoryl-5-methyl-1-pyrroline 1-oxide in 3 ml ofheavy water, D₂ O. The reaction is performed at room temperature underan inert atmosphere (argon) protected from light. The reaction ismonitored by NMR. After 24 hours, the disappearance of the ethylenicproton signal is noted. The reaction medium is then extracted withdichloromethane (3×10 ml) which has been freshly distilled overphosphorus pentoxide (P₂ O₅). The organic phase is then evaporated underreduced pressure. 0.3 g of a residual yellow oil is obtained.

We claim:
 1. A compound of general formula I: ##STR26## in which: R₁represents a phenyl or alternatively a C₁ -C₁₈ alkyl,R₂ represents ahydrogen or deuterium atom, a phenyl or (C₁ -C₁₈) alkyl group or aradical Z of formula ##STR27## in which A is a single bond, a methylenegroup of an oxamethylene group (in which the oxeygen atom is linked tothe phosphorus atom of the radical Z), Y represents an oxygen atom and Rrepresents a hydrogen atom, a (C₁ -C₁₈) alkyl or a (C₆ -C₁₈) aryl, oralternatively Y represents a methylene group and R represents a hydorgenatom, a (C₁ -C₁₇) alkyl or a (C₆ -C₁₈) aryl, R₃, R₄ and R₅ represent,independently of one another, a hydorgen or deuterium atom of a phenylor (C₁ -C₁₈) alkyl group, and R₆ represent a hydrogen or deuterium atom,a phenyl or (C₁ -C₁₈) alkyl group of said radical Z R₇ representing ahydrogen or deuterium atom or a methyl group, with the proviso the oneof the groups R₂ and R₆ must represent said radical Z, whereas the otherof the groups R₂ and R₆ cannot represent said radical Z; as well astheir corresponding physiologically acceptable salts obtained by theaction of an inorganic or organic base.
 2. Compounds according to claim1, of formula II: ##STR28## in which: R represents a hydrogen atom, a(C₁ -C₁₈) alkyl or a (C₆ -C₁₈) aryl,R₁ represents a phenyl oralternatively a (C₁ -C₁₈) alkyl, R₃, R₄, R₅ and R₆ represent,independently of one another, a hydrogen or deuterium atom or a phenylor (C₁ -C₁₈) alkyl group, and R₇ represents a hydrogen or deuterium atomor a methyl group.
 3. Compounds of formula II according to claim 2, inwhich R₁ s a methyl group and R₇ is a hydrogen or deuterium atom. 4.Compound selected from:5-diethoxyphosphoryl-5-methyl-1-pyrroline1-oxide, 5-phosphono-5-methyl-1-pyrroline 1-oxide, and5-diethoxyphosphoryl-5-methyl-(2,3,3-² H₃) -1-pyrroline 1-oxide, as wellas its corresponding physiologically acceptable salts obtained by theaction of an inorganic or organic base.